Blunted blades: new CRISPR-derived technologies to dissect microbial multi-drug resistance and biofilm formation

ABSTRACT The spread of multi-drug-resistant (MDR) pathogens has rapidly outpaced the development of effective treatments. Diverse resistance mechanisms further limit the effectiveness of our best treatments, including multi-drug regimens and last line-of-defense antimicrobials. Biofilm formation is a powerful component of microbial pathogenesis, providing a scaffold for efficient colonization and shielding against anti-microbials, which further complicates drug resistance studies. Early genetic knockout tools didn’t allow the study of essential genes, but clustered regularly interspaced palindromic repeat inference (CRISPRi) technologies have overcome this challenge via genetic silencing. These tools rapidly evolved to meet new demands and exploit native CRISPR systems. Modern tools range from the creation of massive CRISPRi libraries to tunable modulation of gene expression with CRISPR activation (CRISPRa). This review discusses the rapid expansion of CRISPRi/a-based technologies, their use in investigating MDR and biofilm formation, and how this drives further development of a potent tool to comprehensively examine multi-drug resistance.

are repaired by error-prone non-homologous end joining (NHEJ) in eukaryotes, usually disrupting the gene, also known as "knocking out" (KO).While many prokaryotes lack this mechanism of repair, CRISPR-KO through NHEJ is still possible (12,13).Designing synthetic single guide RNA (sgRNA) allows the targeting of nearly any location in a genome, though the site needs to be next to specific nucleotide sequences known as protospacer adjacent motifs (PAM sites) (14)(15)(16).
The first CRISPRi systems incorporated the same "recognize target sequence and guide nuclease for subsequent degradation" design as baseline CRISPR, but with a catalytically dead Cas9 variant (dCas9) (16)(17)(18)(19).Instead of making double-stranded cuts, mutations in both of the Cas9 catalytic domains effectively dull the "molecular scissors." Though unable to cut DNA at the sgRNA target, bound dCas9 sterically hinders transcription initiation or elongation.sgRNA design strategies use this inhibition to allow selective knockdown of nearly any gene, including essential genes (20).When CRISPRi systems are combined with inducible promoters, titratable levels of gene repression are possible, as opposed to the binary "present or completely knocked out" phenotype of CRISPR-KO (21).CRISPRi screens can therefore be truly genome wide, as essential genes can be investigated through tunable knockdown.
CRISPRi built around dCas9 has been further modified through the creation of dCas9 fusion proteins.CRISPRi using dCas9 fused to transcription repressors provides enhanced gene knockdown in some tools (Fig. 1B) (22,23).CRISPR activation (CRISPRa) systems use sgRNA to guide dCas9 fused to transcriptional activators to intended sites, typically upstream of the target gene.Instead of physically blocking transcription, dCas9 positions the fused transcriptional activator near features such as promoter sequences to induce gene expression (Fig. 1B) (19,24).CRISPRa was described as early as 2013, where dCas9 fused with the omega subunit of RNA polymerase led to targeted gene activation in Escherichia coli (19).More recent technologies have expanded this technique to fungal pathogens, utilizing different transcriptional activators (24).
It is important to note that not all CRISPR-derived tools are built around dCas9.CRISPR systems are quite diverse and are categorized as class 1 or class 2, which are further divided into Types I through VI.Class 1 multiple-Cas systems consist of Type I, III, and IV systems, while class 2 systems utilize a single effector Cas protein and include Types II, V, and VI (25,26).An overview of the native mechanisms and the technology derived from them can be seen in Fig. 1.Therefore, CRISPRi/a that utilizes dCas9 is built off a Type II CRISPR system.Its simplicity and well-described nature led to the creation of gene modulation tools in Gram-negative, indeterminate, positive, and fungal species (15,19,22,27,28).
Despite the frequency of class 2 systems in CRISPRi tools, native class 1 systems account for ~90% of identified CRISPR loci (34).This includes the variety of Type I subsystems, Type IA through 1F, many with additional subvariants.These exhibit different arrangements of various Cas proteins, but all feature Cascade, which are multi-Cas complexes that recruit an effector nuclease for DNA degradation (25).Type III systems utilize a complex of Cas10 proteins similar to the Cascade seen in Type I (25).Type III systems target both DNA and RNA, including the crRNA (35).This self-targeting mechanism has likely hindered Type III CRISPRi development (Fig. 1A) (35,36).Interest ingly, Type IV CRISPR systems lack a functional nuclease entirely, and therefore, the Cas complex only sterically hinders transcription, essentially working as a native CRISPRi system (Fig. 1A) (37).
All but Type III systems have been adapted to create CRISPRi technologies, which have continued to evolve as they are leveraged to study pathogenesis across a multitude of species (37)(38)(39)(40).CRISPRa has been developed alongside the earliest CRISPRi tools and has been successfully utilized in some pathogens.Therefore, this review will discuss how the versatility of CRISPRi/a technologies has aided in the identification and validation of conditionally essential genes within pathogenic species that promote drug resistance and biofilm formation (Table 1) as well as how these studies have contributed to the expansion of available CRISPRi-based tools.

MULTI-DRUG RESISTANCE
Both bacteria and fungi show great plasticity in surviving antimicrobial treatments.Importantly, MDR organisms have outcompeted our capacity to produce new antibiot ics or therapies to treat infections, especially the clinically relevant ESKAPE pathogens (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter bauman nii, Pseudomonas aeruginosa, and Enterobacter spp.) (69).Therefore, understanding the role of essential pathways involved in antimicrobial resistance is crucial to develop efficient therapies against Gram-negative, indeterminate, and positive bacterial and fungal species.

Escherichia coli
Escherichia coli is among the most common nosocomial pathogens, especially in urinary tract infections.These bacteria often possess antibiotic resistance via β-lactamases, acquired though horizontal gene transfer (HGT).This has led to additional carbapenem resistance among some nosocomial E. coli strains (70).Acquisition of drug resistance genes from a diverse range of pathogenic bacteria can be attributable to mobile genetic elements, including Class 1 integrons (71).Due to the genetic mobility and diversity of carried resistance genes, Class 1 integrons are difficult to target for elimination.Utilization of CRISPRi has proven successful in inhibiting them in E. coli, decreasing the expression of integrase (intI1) by ~96% which resulted in 1,000-fold reduction in HGT rate (41).Furthermore, multiple sgRNAs were designed to target the dfrB2 cassette and sul1, which encode a dihydrofolate reductase and a dihydropteroate synthase.These promote resistance to trimethoprim and sulfamethoxazole, respectively (41,72,73).
One mechanism of MDR in E. coli is to express efflux pumps like AcrAB-TolC.This pump provides resistance to multiple antibiotics, including carbapenems, erythromycin, tetracycline, and rifampicin (74,75).AcrAB-TolC consists of the outer membrane channel, TolC, which is connected by AcrA to the inner membrane transporter AcrB (76).Using an engineered CRISPRi system, acrA, acrB, and tolC were targeted independently or simultaneously by using plasmids containing clustered sgRNAs.Single guide targeting of acrA lead to downregulation of acrB and vice versa, while neither reduced tolC expres sion.Given the location of acrA and acrB within their operon, this was unsurprising.Interestingly, single guides targeting tolC did reduce expression of acrA and acrB.By clustering sgRNA sequences, simultaneous targeting of each gene was achieved.Both single and simultaneous silencing of the efflux pump genes increased susceptibility to rifampicin, erythromycin, and tetracycline, with simultaneous targeting outperforming single sgRNA plasmids (42).These findings indicate that single-target silencing can cause repression in other genes within the studied system, while targeting multiple genes can enhance overall repression.
A potential complication for CRISPRi technology is the presence of multi-copy resistance genes on natural plasmids, especially in clinical strains.The Yao group engineered an all-in-one CRISPRi system to overcome this issue (43).This system was tested in the clinical E. coli strain E0171, which contains two copies of the carbapenemase gene bla KPC-2 .Using a single sgRNA that targets the non-template strand of its coding region, they were able to target multi-copy bla KPC-2 to reduce expression 600-fold, making E. coli susceptible to meropenem (43).

Pseudomonas aeruginosa
Development of genetic tools for P. aeruginosa has proven difficult, particularly among MDR strains.Some laboratory strains permit the use of Type II dCas9 tools, like PAO1.The unidentified essential gene PA0715 was characterized in PAO1 using CRISPRi, revealing a role in amino acid, carbohydrate, ketone body, and organic salt metabolic pathways, as well as motility.After PA0715 silencing, PAO1 was sensitized to sample antibiotics from penicillin, third-generation quinolone, aminoglycoside, and macrolide antibiotics, suggesting it presents a potential new antibiotic target (46).Genetic tool development is complicated by native Type IF CRISPR systems, which are found in up to 30% of P. aeruginosa strains (77).These target foreign DNA, including engineered CRISPRi constructs (45,77,78).Xu and collaborators demonstrated that these native systems can be repurposed, using the MDR strain PA154197.After modifying the native CRISPR, they used it to target the efflux pumps MexAB-OprM, MexEF-OprN, and MexGHI-OpmD by knocking out the components mexB, mexF, and mexH, resulting in increased sensitivity to fluoroquinolones and beta-lactams (45).Although this work used CRISPR-KO, it demonstrated successful modification of native CRISPR systems in P. aeruginosa clinical isolates (78).
Chen and collaborators created a CRISPRi tool by removing the cas2-3 gene from Type IF CRISPR that integrates at the attB site in the P. aeruginosa chromosome (78).This design allows genetic silencing in clinical MDR strains, where intrinsic resistances make selection-based plasmid retention difficult (78).This system is specific to P. aeruginosa, and strains with functional native Type IF CRISPRs will eventually target and eliminate the foreign CRISPRi sequence (38).To overcome this challenge, a novel plasmid-based CRISPRi platform (CSYi) was created.To protect against native CRISPR targeting and destruction, they added two anti-CRISPR proteins AcrlF3 and AcrlF23 to the plasmid (38).This system was successfully validated by repressing expression of CzcR, a zinc regulator, by ~50% (38).CzcR modulates multi-drug resistance in Pseudomonas through repres sion of OprD, a porin responsible for carbapenem uptake, and both MexAB-OprM and MexGHI-OpmD efflux pumps (38).CzcR repression sensitized Pseudomonas to levofloxacin, proving its role in carbapenem resistance (38).Importantly, this plasmid is not limited to Pseudomonas, as it was also shown to work in E. coli strains (38).

Klebsiella pneumoniae
Type I CRISPR loci are prevalent in MDR isolates of the ESKAPE pathogen K. pneumoniae, which also frequently harbor multiple resistance genes on large plasmids (79).Through one-step cloning, an inducible CRISPRi system was developed to silence multiple resistance genes in pNK01067-NDM-1, a natural MDR plasmid in a K. pneumoniae isolate.
Single-target silencing of bla NDM-1 reduced meropenem resistance by ~1,000-fold and modifications to the CRISPRi design allowed targeting of both bla NDM-1 and bla SHV-12 on the large MDR plasmid, making the isolate susceptible to meropenem and aztreonam.Importantly, gene silencing on the scale of an entire plasmid operon was also possible, by targeting the promoter region of bla NDM-1, which silenced the bla NDM-1 -ble MBL -trpF operon (43).Mobile-CRISPRi in K. pneumoniae has been successful.This system uses Type II dCas9 CRISPRi built for ease of library construction, conjugative transfer across species, and stable genomic integration.Using this system, the K. pneumoniae essential gene folA was targeted.This gene encodes dihydrofolate reductase and is the target of trimetho prim.Complete folA gene silencing was lethal, while partial knockdown sensitized K. pneumoniae to the drug (49).
This system has also proven useful in a screening strategy similar to Tn-Seq, to assess which genes or pathways have vital roles in antimicrobial resistance.Mobile-CRISPRi-Seq was applied to 870 predicted essential genes under antibiotic pressures at 25% MIC, which identified folB and folP as trimethoprim resistance genes (50).Additional genes of interest include waaE, a component of LPS biosynthesis and polymyxin B resistance, and fldA, an electron transport carrier and contributor to beta-lactam resistance (50).

Acinetobacter baumannii
A. baumannii is an emerging MDR nosocomial pathogen, and its treatment is increasingly challenging (80).To understand A. baumannii mechanisms of drug resistance, a CRISPRi library was created and screened using last-resort antibiotics including fosfomycin, colistin, imipenem, meropenem, and rifampicin (51).Knockdown of cell wall assembly, peptidoglycan synthesis, and translocation genes (ftsI, murA, dapA, and murJ) resulted in increased sensitivity to imipenem and meropenem (51).Interestingly, treatment of A. baumannii with fosfomycin-which targets the product of murA-was ineffective due to efflux pump activity.However, when fosfomycin and imipenem were administered in tandem, the two exhibited synergistic effects (51).Further antibiotic-gene interaction analysis showed that colistin and rifampicin have strong opposite phenotypes for genes encoding NADH dehydrogenase complex I (NDH-1) and lipooligosaccharide.CRISPRi knockdown of NDH-1 function recapitulated the reduced membrane permeability found in rifampicin resistance.This sensitizes A. baumannii to colistin, which inhibits NDH-2.Importantly, these results indicate how anticorrelated phenotypes have been unveiled due to CRISPRi, to better exploit synergistic antibacterial treatment (51).

Mycobacterium spp.
Given the global prevalence and severity of Mycobacterium tuberculosis (Mtb), it is not surprising that the CRISPRi technology has been quickly added to the arsenal that aids in understanding the mechanisms of mycobacterial MDR (81).In the Mtb field, CRISPRi is a powerful tool for examining essential genes and has also been effective in conjunction with KO libraries, including CRISPR-KO and Tn-seq libraries (52).When used in conjunction with KO libraries, essential genes identified by CRISPRi can be further explored to determine whether a polar effect contributes to their essentiality (52).Using this strategy, the Sun lab found 29 essential genes that impacted resistance or suscepti bility to the antimycobacterial bedaquiline (BDQ), which inhibits the mycobacterial ATP synthase (52).This was achieved by growing both CRISPR-KO and CRISPRi libraries to late-log phase, mimicking in vivo mycobacterial growth conditions before adding BDQ (52).While both libraries detected genes resistant and susceptible to BDQ treatment, 29 essential genes were only identifiable by CRISPRi.One discovery included the drug efflux pump component mmpSL5 (52).The absence of this gene resulted in BDQ sensitivity while inactivation of mmpSL5's repressor, rv0678, led to acquired BDQ resistance by overexpressing the efflux pump (52).Additionally, they found that many essential genes involved in ATP synthesis such as atpB, atpC, and atpH are potential synergistic targets for antimicrobial effects.Another potential synergistic gene only detectable by CRISPRi in this study was pks13 (52).Pks13 is an essential enzyme that forms mycolic acids and previously was found to be the target of the antimicrobial drug, TAM16 (82).This screen also unveiled unpredicted factors to play a role in drug-gene interactions such as Topoisomerase I (topA).The topA depletion increased BDQ sensitivity, suggesting that Topoisomerase I may indirectly affect ATP homeostasis (52).
Bosch and colleagues utilized a titratable CRISPRi platform to measure genetic vulnerability in Mtb, including that of a hypervirulent isolate.They constructed a CRISPRi library against 98.2% of all annotated Mtb genes.Instead of inducible sgRNA, this system used Sth1dCas9 instead of dCas9, which allows for a gradient of gene knockdown through recognition of non-canonical PAM sites.The estimated sgRNA strength was then used to predict the degree of gene knockdown, which was used in conjunction with the observed fitness cost in the final model of genetic vulnerability.This process was validated in Mycobacterium smegmatis and resulted in a quantitative vulnerability index for ~93% of all TnSeq essential Mtb genes (28).Pathway enrichment of the most vulnerable genes revealed protein synthesis and tRNA synthesis were vulnerable targets, while amino acid biosynthesis was not.The Clp protease complex was found among the most vulnerable gene set, validating active interest in antimycobacterials targeting the protease (28,83).In contrast, peptide deformalyse def is an essential Mtb gene previously suggested as a potential antimycobacterial target (84).The genome-wide CRISPRi assessment revealed def as highly invulnerable, suggesting that some essential genes are not good candidates for successful drug development (28).
A CRISPRi chemical genetics platform has been used to titer the expression of Mtb genes (53).By utilizing this approach with multiple antimicrobials, Li and collabo rators uncovered multiple mechanisms of acquired resistance, including the response regulator mtrA.Knockdown of mtrA sensitized Mtb to rifampicin, vancomycin, and BDQ and impaired intracellular survival within macrophages (53).Ethidium bromide and a fluorescent vancomycin conjugate both showed that mtrA knockdown leads to increased cell envelope permeability (53).
Tuberculosis antibiotic regimens do not include the use of beta-lactams; however, a combinatory therapy with β-lactamase inhibitors is a prospective strategy to treat MDR Mtb (54).To dissect whether peptidoglycan modifications could promote synergistic effects with antimicrobial resistance and intracellular survival, Silveiro and collaborators CRISPRi silenced namH and murT/gatD.These encode enzymes responsible for the peptidoglycan modifications D-iso-glutamate amidation and N-glycosylation of muramic acid, respectively (54).By using CRISPRi silencing in M. smegmatis strains, which lack the β-lactamase (blaS), they found that namH was essential to mycobacterial survival while murT/gatD was dispensable.Furthermore, silencing of namH affected cefotaxime and isoniazid resistance and silencing murT/gatD reduced resistance to β-lactams.Further more, simultaneous depletion of both genes not only resulted in synergistic increasing of beta-lactam susceptibility but also significantly promoted killing by macrophages (54).As these modifications were highly conserved in a set of 172 clinical tuberculosis strains, these results suggest that peptidoglycan modifications contribute to pathogenicity in Mtb and could be a potential therapeutic target (54).
Recent studies have shown that Mtb dedicates great effort to produce, sense, and degrade cyclic AMP (cAMP) (55,(85)(86)(87), suggesting that this molecule may play a role in Mtb physiology.Interestingly, there is only one Mtb-essential adenylate cyclase (rv3645) that catalyzes the conversion of ATP to cAMP (55).To dissect the potential roles of cAMP in Mtb, this adenylate cyclase was silenced by CRISPRi, showing that the lack of cAMP increased Mtb sensitivity to vancomycin, rifampicin, and clarithromycin (55).Furthermore, cAMP reduction inhibited Mtb growth in the presence of long-chain fatty acids, a host-relevant carbon source.This study's finding that cAMP signaling is important for MDR and fatty acid metabolism suggests that its disruption may provide another avenue for Mtb management (55).
CRISPRi chemical genetics identified that loss of function of the essential gene rv2477c in Mtb confers resistance to streptomycin, amikacin, ethambutol, rifampicin, and levoflozacin (53).This gene is an ortholog of the E. coli gene ettA, which is involved in the translation elongation cycle; however, it is not essential in E. coli (88,89).Interest ingly, silencing of the ettA homolog in M. smegmatis resulted in upregulation of two proteins, HflX and Eis, which are part of the whiB7 stress response regulon in Mtb (53).Based on these data, it was hypothesized that whiB7 upregulation may contribute to acquired drug resistance.Depletion of both whiB7 and ettA in Mtb reversed aminoglyco side resistance.However, whiB7 knockdown did not reverse ethambutol or levofloxacin resistance, suggesting the mechanism of resistance is whiB7 independent (53).
Even though Mycobacterium abscessus is not closely related to Mtb, it causes debilitating TB-like pulmonary infections and is highly drug resistant, limiting treatment options (90).Understanding M. abscessus pathophysiology has been hindered by a lack of genetic tools.Recently, a mycobacterial single-plasmid CRISPRi-dCas9 system optimized for inducible gene silencing in Mtb and M. smegmatis was evaluated in M. abscessus (56,91).To test this platform, the authors targeted two well-characterized antimicrobial resistance genes, bla and whiB7, which are responsible for beta-lactam and macrolide resistance in M. abscessus, respectively (92,93).Depletion of these genes resulted in antimicrobial sensitivity, thereby validating the platform.Following these results, they targeted the essential genes ftsZ and topA in M. abscessus, where the first encodes for the cell division protein FtsZ and the second encodes topoisomerase I.These two genes are also essential in Mtb and M. smegmatis and are attractive drug targets (94,95).
These studies have shown that CRISPRi identification of novel gene pathways involved in multi-drug resistance may be critical to generate successful solutions against the prevalence of MDR Mtb and other mycobacterial strains.

Enterococcus faecalis
Native CRISPR loci have been frequently described in E. faecalis strains, which hinder foreign DNA acquisition, including resistance plasmids (96,97).Pathogenic E. faecalis strains largely lack functional CRISPR loci, a finding replicated in vivo with commensal E. faecalis in the mouse intestine (96).One such example is the vancomycin-resistant strain V583, which lost a functional CRISPR system while obtaining antibiotic resistance (97)(98)(99).Thus, CRISPRi systems may present an advantageous tool for the examination of MDR in E. faecalis clinical isolates.CRISPRi has already been demonstrated in the E. faecalis strain OG1RF, an oral infection isolate that possesses one functional native CRISPR system (97).For example, the Kline group developed a multiplex system around streptococcal CRISPRi.They first showed that streptococcal crRNA is not recognized by enterococcal dCas9, preventing interference from the native CRISPR systems of E. faecalis strains (15).They then silenced croR, which is part of the CroRS two-component system that contributes to antibiotic resistance and intracellular survival within macrophages.croR knockdown reduced bacitracin resistance (15), showing similar results to a croR transposon-mediated knockout (100).

Clostridioides difficile
The high prevalence of MDR Clostridioides difficile in both community-and hospitalacquired infections has become worrisome due to limited treatment options (101).The frequent use of multiple antibiotics depletes native microfauna in patients' GI tracts, providing a niche for MDR C. difficile to exploit (102).C. difficile has species-specific features such as its lipid membrane, which lacks phosphatidylserine and is ~50% glycolipid in composition (57).A titratable CRISPRi system was developed for C. difficile to better understand cell wall synthesis, identifying the previously uncharacter ized peptidoglycan synthase PBP-0712, which is important for proper elongation, cell division, and protection against lysis (21).Using CRISPRi, it was further that the two-component system HexRK was essential for antibiotic resistance.This target was initially identified through a transposon insertion library, with a relatively undescri bed B. subtilis homolog.Silencing of the HexRK operon (hexSDF) reduced daptomycin and bacitracin resistance by fourfold and eliminated the unique C. difficile glycoli pid HNHDRG, which likely contributes to this resistance (57).Silencing of individual HexRK operon genes suggested specific roles for producing HNHDRG, which normally comprises ~16% of the bacterial membrane.HexS and HexD are required for the production of a glycolipid intermediate and its conversion to HNHDRG, respectively, while HexF is not essential (57).As HNHDRG is only found in C. difficile, this suggests that CRISPRi silencing of unique features permits deeper dissection of novel pathways, potentially aiding the identification of species-specific druggable targets.

Staphylococcus spp.
Staphylococcus aureus is a highly prevalent nosocomial pathogen and possesses resistance strategies against beta-lactams, linezolid, daptomycin, vancomycin, fluoroquinolones, and tetracycline (103).Among the identified mechanisms of antibiotic resistance are efflux pumps and peptidoglycan modification, many of which are the result of direct changes to chromosomal genes via spontaneous mutation or HGT (104).Due to both intrinsic and acquired antimicrobial resistance, treatment of MDR S. aureus is extremely challenging (105).To address this challenge, multiple novel CRISPRi systems capable of multi-gene silencing in S. aureus have been developed (58,106).
For example, Zhao and collaborators utilized an anhydrotetracycline (ATc)-inducible promoter to allow inducible and reversible repression of multiple genes.Simultaneous targeting of virulence factors including hemolysin (hla) and staphylococcal protein A (spa) validated its multi-gene knockdown activity.Using this new system, they were also able to reverse hla silencing to baseline expression after removal of ATc.Furthermore, they also silenced mecA, a beta-lactam resistance gene in methicillin-resistant S. aureus (MRSA) strain N315, sensitizing the MDR strain to oxacillin (58).
Another novel CRISPRi system is pBACi, which successfully silenced a plasmid-bound resistance gene within in a clinical isolate (59).Genetically recalcitrant clinical S. aureus isolates possess restriction enzymes that inhibit retention of the conventional vector.To overcome this limitation, pBACi was constructed in B strain E. coli, which lacks the components targeted by the S. aureus restriction enzymes.Validation of pBACi was done by silencing the β-lactamase gene blaZ, which significantly reduced both mRNA levels and β-lactamase activity by ~50% (59).
To perform unbiased whole genome screening, two CRISPRi libraries were created in S. aureus strains RN4220 and NCTC8325.The RN4220 library comprised all 2,666 annotated genes and NCTC8325 covered 2,836 genes (20,60).Screening of the RN4220 library provided evidence that ctaABM, mnhABCDEFG, and ndh are involved in aminogly coside resistance mechanisms (60).These genes are involved in pathways that lead into the electron transport chain (ETC), which is consistent with the current understanding that disruption of the ETC alters membrane potential and lowers aminoglycoside uptake (60).Additionally, the essential genes in the mevalonate pathway, mvaD, mvaK2, and Topoisomerase I (topA), were also revealed to be novel loci in aminoglycoside sensi tivity (60).Furthermore, transcriptional inhibition of several essential ribosomal genes provided gentamicin tolerance, suggesting that ribosomal perturbation may provide some defense against aminoglycosides (60).
The NCTC8325 CRISPRi library was screened against dalbavancin, identifying several genes that confer drug resistance or susceptibility.These genes were involved in cell wall modification (dltABCD and pbp4), ABC-transporter (vraFG), cell division (ezrA), ribosomal operon (rpsF-ssb-rpsR), and biosynthesis of deoxyribonucleotides (nrdF).Additionally, the uncharacterized genes SAOUHSC_00678 and SAOUHSC_00892 (a putative RNA-binding protein) were found to be involved in antimicrobial susceptibility.Interestingly, silencing the lipoprotein KapB, a non-essential gene (kapB), only confers resistance to dalbavan cin and suggests a unique mechanism of action (20).Finally, this library was used to demonstrate that dalbavancin tolerance is modulated by the metabolic Shikimate pathway, through knockdown of the sagB, aroB, and vrfA genes (20).
Treatment of methicillin-resistant S. aureus (MRSA) is often heavily reliant on lastresort antibiotics like vancomycin.When such treatment fails, it is usually due to intermediate vancomycin resistance due to cell wall thickening.By examining RNA-RNA interactions, Mediati and collaborators identified a regulatory region within the 3′UTR of vigR mRNA.This vigR 3′UTR enhanced isaA expression, which is a cell wall lytic transgly cosylase (61).Using CRISPRi, vigR mRNA expression was reduced, resulting in 1000-fold susceptibility to sub-inhibitory vancomycin concentrations.Knockdown of isaA revealed reduction in cell wall thickness, which partially re-sensitized S. aureus to vancomycin.This effect was less successful CRISPRi inhibition of vigR 3′UTR, which may encourage further CRISPRi use in non-coding genetic regions (61).

Saccharomyces cerevisiae
Extending CRISPRi systems to fungal species has faced additional challenges than those present in prokaryotes.For example, diploid fungi pose a barrier in creation of CRISPR-KO strains.Despite such challenges, a plasmid-based, ATc-inducible CRISPRi system was described in the yeast Saccharomyces cerevisiae.This yeast CRISPRi system included a dCas9 with the Mxi1 transcriptional repressor fused to the C-terminus of dCas9 (Fig. 1B).This system reduced expression of the targeted gene, and the transcription start site (TSS) and 200 bp upstream of the TSS were identified as the best target site for dCas9 fusion proteins.This technology also uncovered that repression of the C-4 methyl sterol oxidase, Erg25, promoted resistance to fluconazole (23).

Candida albicans
Candida spp.lack a native, autonomously replicating plasmid system and have limited selection targets for plasmid maintenance, presenting an additional barrier to the creation of CRISPRi/a tools.CRISPR-KO tools built around codon-optimized Cas9 were successfully demonstrated in 2015, though essential gene functions were unable to be addressed (107).The first functioning CRISPRi system in C. albicans was published in 2019, using two dCas9 fusion constructions, dCas9-Mxi1 and dCas9-Mig1 (22).Both Mxi1 and Mig1 are transcriptional repressors that showed higher silencing efficacy when working together.Contrary to the earlier S. cerevisiae system, this CRISPRi construct utilized C. albicans NEUT5L homology regions for plasmid integration.Silencing the essential chaperon HSP90 (ADE2) validated this system (22).This study showed that like in S. cerevisiae, HSP90 also contributed to C. albicans fluconazole resistance (22,108).
Another CRISPR technology recently developed for C. albicans allows the modulation of gene expression (109).This system was created by fusing the reporter gene gfp to the cytosolic catalase CAT1 promoter as part of an integrative plasmid containing the CRISPRi system.This initial system maintained sgRNA expression under the constitutive SNR52 promoter but was modified by utilizing catalytically dead dCas9 under a tetracyclineinducible promoter to allow for both genetic upregulation and downregulation.Its silencing capabilities were validated by targeting gfp, reducing expression by ~30%.The silencing capacity was further improved by fusing the hyphal gene repressor Nrg1 to dCas9, reducing gfp expression by 45% (109).CRISPR activation (CRISPRa) was developed by exchanging Nrg1 for the transcriptional activator Gal4, showing a twofold increase in gfp expression.This was further enhanced by adding a second fusion protein, MCP-V64.The presence of both Gal4 and VP64 transcriptional activators significantly enhanced gfp expression (109).
The CRISPRa system was further developed in C. albicans to examine antifungal susceptibility (24).This utilized a fusion protein consisting of dCas9 and the tripartite activator complex VPR.Antifungal resistance was examined with the genes CDR1 and YAK1.CDR1 is an efflux pump gene associated with azole resistance, and CRISPRa upregulation enhanced transcription by ~2.9-to 5.6-fold resulting in reduced sensitiv ity to fluconazole.YAK1 is a kinase whose overexpression promotes filamentation and resistance to the antifungal amphotericin B (110,111).Using this CRISPRa system, YAK1 expression was doubled, increasing amphotericin B resistance (24).

Nakaseomyces glabrata
Formerly considered a member of the Candida genus, Nakaseomyces glabrata is the second most common cause of candidiasis infection.Clinical isolates have increas ingly demonstrated multi-drug resistance, including against azole and echinocandin antifungals (112,113).Both CRISPRi and CRISPRa have proven useful in the study of N. glabrata, taking advantage of dCas9 fusion protein strategies.Using a dCas9-Mxi1 fusion protein, a new CRISPRi system was created and validated via URA3 silencing (64).Since it was known that ALG3 deletion in S. cerevisiae promotes resistance to the toxin HM-1, this system was tested by silencing the unverified ALG3 in N. glabrata, which proved that reduction of ALG3 expression promotes fungal growth in presence of the toxin (64,114).
Recently, CRISPRa utilizing dCas9-VPR has been described in N. glabrata.The transcription regulator PDR1 was first targeted, with 10 unique sgRNAs created due to differences in the reported TSS of PDR1.The two sgRNA targeting ~550 bp upstream of the start codon led to a 1.5-fold increase in PDR1 transcription and subsequent reduction in fluconazole sensitivity (65).This further emphasizes the importance of TSS proximity for successful sgRNA design.This tool was subsequently used to investigate STE11 and SLT2 due to their possible roles in drug or environmental resistances.STE11 mediates tolerance to environmental challenges such as oxidative stress, and SLT2 is overexpressed when N. glabrata is treated with the antifungal caspofungin (115).CRISPRa overexpres sion of STE11 was achieved as a 1.2-to 1.8-fold increase and conferred tolerance to H 2 O 2 -induced oxidative stress while SLT2 was overexpressed 1.8-to 2.8-fold and led to greater N. glabrata fitness in the presence of caspofungin (65).

BIOFILMS
Nosocomial infections frequently involve biofilms on tissue and medical devices (6)(7)(8).Biofilms are often associated with antibiotic resistance, promoting chronic and persistent infections, which make them difficult to treat (6)(7)(8).Microbial biofilm formation is complex, involving signaling and regulatory pathways that control the transition from motile to sessile lifestyle, metabolic status, cell coordination, production of an extracel lular polymeric matrix, and maturation of the biofilm's 3D structure.Therefore, under standing the intricacies of biofilm formation is critical for developing treatments against infections.

E. coli
E. coli biofilms are prevalent on medical devices, contributing to their multi-drug resistance (3,116).Previous reports have shown efflux pump activity is associated with biofilm formation in A. baumannii, P. aeruginosa, and S. enterica serotype Typhimurium (117)(118)(119).CRISPRi targeting of the AcrAB-TolC efflux pump system in E. coli inhibited biofilm formation by ~50% in addition to sensitizing to rifampicin, erythromycin, and tetracycline (42).Transcription of acrA, acrB, and tolC was correlated with drug MICs while acrB and tolC correlated with biofilm formation, though their direct or indirect contributions to biofilm formation remain unclear.
Quorum sensing (QS) is another mechanism important for biofilm formation in E. coli.This is facilitated through coordination of cell-cell signaling by producing and detecting autoinducers (AIs) to transcriptionally synchronize behaviors including virulence, biofilm formation, and survival (120)(121)(122).Therefore, targeting QS systems may help dissect steps of biofilm formation.The Khan group targeted the luxS gene, encoding the synthase of the AI-2 QS molecule, which is critical during the initial stages of biofilm formation (27,123).This group developed a plasmid-based CRISPRi system to knock down luxS in the E. coli clinical strain AK-117.Compared with controls, the CRISPRi-strain exhibited ~95% reduction in luxS expression, resulting in ~50% reduction in biofilm formation (27).

Pseudomonas spp.
Pseudomonas spp.employ different strategies of environmental persistence including biofilm formation, motility, and production of virulence factors (124,125).To further understand the mechanisms of Pseudomonas spp.pathogenesis and biofilm formation, CRISPRi has been used to elucidate the role of uncharacterized essential genes.For example, the novel, unidentified essential gene PA0715 in the strain PAO1 was targeted by Zhou and collaborators.Its downregulation resulted in reduced growth rate, motility, chemotaxis, antibiotic resistance, pyocyanin production, and biofilm formation (46).To examine the role of PA0715 in virulence, CRISPRi-silenced and control strains were tested in Galleria mellonella larvae, finding that downregulation significantly reduced virulence (46).These findings support transcriptomic analyses that showed PA0715 may play a role as a global regulator that influences metabolic signaling pathways affecting motility, biofilm formation, and virulence.While the mechanisms by which PA0715 regulates virulence and biofilm formation are not described, this study paves the way to dissect the role of uncharacterized essential genes.
Additional studies in the non-pathogenic P. fluorescens unveiled genes that control biofilm formation.Using three diverse P. fluorescens strains, SBW25, WH6, and Pf0-1, Noirot-Gros and collaborators silenced genes involved in the GacA/S two-component system (gacS) and regulatory proteins involved in cyclic di-GMP signaling (bifA, dipA, and rimA) (47).They found that silencing of gacS, rimA, bifA, and dipA resulted in reduced swarming abilities, affecting biofilm thickness and roughness in varying degrees.The authors suggest that the variability in effects may be due to silencing genes at the beginning of or within operons (47).

E. faecalis
Biofilms provide E. faecalis a mechanism to colonize inhospitable conditions and, in some cases, include other species in a polymicrobial community.As we previously mentioned, biofilm formation is a very dynamic process, with the potential for genes to become essential at specific stages of development.Afonina and collaborators demonstrated the utility of inducible CRISPRi to study E. faecalis genes involved in biofilms in a stage-spe cific manner (15).The authors targeted the endocarditis-and biofilm-associated pili (Ebp) that plays a key role in E. faecalis attachment and biofilm formation (126,127).By targeting members of the pilus or the whole ebpABC operon, the authors confirmed the importance of the pili in the initial stage of biofilm formation.Furthermore, the role of Ebp pili in biofilm maintenance was tested by silencing the operon after a biofilm formation of 2, 16, or 24 hours, using a nisin-inducible CRISPRi system.All biofilms were incubated for 24 hours post-induction, with Ebp pilus silencing significantly reducing biofilm structure, ultimately indicating that this pilus is important for both biofilm initiation and biofilm maintenance (15).This study shows that nisin could penetrate the entire biofilm and this novel system opens new research venues to investigate the role of conditionally essential genes in a stage-specific biofilm-dependent manner (15).

C. albicans
The study of biofilm formation in C. albicans has been greatly aided by CRISPRa systems.A dCas9-VPR fusion protein was used to investigate the biofilm-relevant adhesins ALS3 and ALS1.The extent of CRISPRa upregulation of ALS3 varied with fungal growth conditions.Overexpression of ALS1 induced robust biofilms; however, excessive upregulation was detrimental to biofilm formation (24).ALS1 overexpression also enhanced biofilm formation in human urine with fibrinogen, a relevant condition for catheter-associated urinary tract infection (CAUTI) (24).This system was subsequently used in a mouse model of CAUTI, revealing that ALS1 is critical to biofilm formation and pathogenesis during CAUTI (63).

N. glabrata
N. glabrata makes use of biofilms for pathogenesis, similar to Candida species (128).Upregulation of the transcription factor EFG1 is known to enhance biofilm growth, and this gene has a well-defined TSS.Multiple guide sgRNAs were created, targeting within −462 and +112 bp relative to the TSS.EFG1 was overexpressed 3.6-to 8.1-fold, and three sgRNAs showed significantly enhanced biofilm forming ability.Each of the three sgRNA targets were within 200 bp upstream of the TSS (−36 bp, −105 bp, and −196 bp), with −105 bp providing the greatest increase in EFG1 expression.Notably, the CRISPRa construct does repress EFG1 if the sgRNA targets within the coding sequence.The authors posit that this may be due to steric inference with RNA polymerase II by the dCas9 fusion protein (65).

Other filamentous fungi
While not all filamentous fungi form complex biofilms, there has been an explosion in the number of filamentous species with developing CRISPRi/a tools in just the last 3 years.In 2021, a CRISPRa vector utilizing a dCas9-VPR transcription activator was described in Penicillium rubens.This system targeted the dCas9-VPR complex to the promoter of macR, a transcription factor that activates a natively transcriptionally silent gene cluster to initiate macrophorin biosynthesis, an antimicrobial compound (66).In 2022, CRISPRi identified the Aspergillus niger gene DAC1, a GlcNAc-6-phosphate deacetylase, as controlling spore-like propagule formation, a phenotypic change also associated with drug resistance (67).Finally, in 2023, multiple gene targets within the filamentous fungal rice pathogens Magnaporthe oryzae and Ustilaginoidea virens were successfully silenced (68).

OUTLOOK
Nobel-winning work that repurposed the bacterial CRISPR-Cas9 system for genomic editing was described in 2012 (10).Within 1 year, the beginnings of CRISPRi were published utilizing a catalytically dead Cas9 (16,18).The subsequent decade has seen rapid development of novel gene modulation technologies, with initial applications characterizing essential genes.CRISPRi/a technologies have been developed for a variety of species (Fig. 2B) to better understand mechanisms of MDR and biofilm formation (Fig. 2A).
The use of these gene expression tools in pathogenic species has faced new chal lenges when compared with genetic editing techniques.The continuous maintenance of CRISPRi systems and design principles for sgRNA have become increasingly sophistica ted, as they address two of the greatest challenges to utilizing these technologies.
Retention of CRISPRi platforms must contend with the limited options for selective pressure in highly drug-resistant isolates, as well as native CRISPR systems that target introduced CRISPRi (38,78).Platforms like pBACi overcome native CRISPRs by including anti-CRISPR proteins, while other tools are built by editing a native system to function as CRISPRi (59,78).Integrative plasmids can eliminate the need for continual selective pressure; the Mobile-CRISPRi platform takes advantage of this to provide stable gene silencing for over 50 generations in every ESKAPE pathogen (49).
Not all sgRNA sequences are created equal, as the degree of gene silencing will vary by sgRNA sequence.This can be exploited to tune the magnitude of genetic knockdown but can also result in lethal fitness loss (44,48,129).Early genome-wide screens in E. coli identified specific, five-nucleotide-long sequences within "bad seed" sgRNA that led to toxic, off-target dCas9 activity, exacerbated by high dCas9 expression (44).Some "bad seed" sgRNA sequences have been identified in E. coli and proven similarly lethal in other species.However, as some "bad seed" sgRNAs only cause dCas9 toxicity in certain species, this indicates that our knowledge of "bad seed" sequences is incomplete (129,130).
The risk of ineffective or "bad seed" sgRNA is a constant one for genome-wide screens, especially as most such libraries use 10 or less functional sgRNA per gene.This risk grows as CRISPRi libraries are constructed for less-studied pathogens and isolates, as well as CRISPRi-Seq libraries that benefit from extensive sgRNA coverage of genes.One technology that may prove useful here is CRISPR adaptation-mediated library manufac turing (CALM) (60).CALM adapts a hyperactive Type II CRISPR-Cas machinery for the creation of sgRNA to target a provided genome.CALM was able to generate hundreds of thousands of unique crRNAs for a 95% coverage of all targetable genomic sites in S. aureus and manufactured an average of more than 100 unique sgRNA per gene in E. coli (60).
CRISPRi/a technologies have also expanded our understanding of essential genes.Genome-wide screens have shown that not all essential genes are vulnerable drug targets nor does in vitro essentiality always equate to pathogenic relevance (28,62).CRISPRi/a shows great promise at identifying genes essential to pathogenesis in vivo.CRISPRa in C. albicans identified Als1 as crucial for pathogenesis in a murine CAUTI model (63).CRISPRi has also been used in murine pneumonia models, which identified essential genes to virulence: exsA in P. aeruginosa; pal, yciS, and rib in K. pneumoniae; and purA in Streptococcus pneumoniae (48,50,62).Tailoring promoter strength for constitutive knockdown offers an alternative to induced CRISPRi, alleviating concerns over tissue penetration by some inducing agents (48).Notably Mobile-CRISPRi-Seq showed that the S. pneumoniae gene metK is dispensable in vivo, despite being essential in vitro (62).Identifying novel genes and pathways conditionally essential to pathogenesis in vivo may offer new insights into drug discovery targets.
CRISPRi/a technologies have revolutionized gene modulation tools over the past decade.The earliest systems developed as Type II dCas9 systems, and these are still utilized as the backbone of many modern tools.In the past few years, new technologies for gene knockdown have been designed utilizing Type I, IV, V, and VI systems (31)(32)(33)(37)(38)(39)(40).It is likely inevitable that CRISPRa techniques will be expanded to include some of these systems as well, taking advantage of the unique mechanisms across CRISPR types.CRISPRi/a technologies have ushered in a new era of antimicrobial research and are invaluable assets in the investigation of complex pathogenic network interactions across bacterial and fungal species, where multi-drug resistance and biofilm formation still pose significant challenges.

FIG 1
FIG 1 Native CRISPR systems and their derived technologies.CRISPR systems are diverse but can be sorted into two classes.Class 1 systems (A) involve multi-Cas complexes whereas class 2 systems (B) require a single nuclease.There are three types of systems in each class, and all but one has been utilized in CRISPRi technology.Cas nuclease and complexes are guided by crRNA (red) to either host DNA (blue) or host RNA, usually mRNA transcripts (black).Figure was created using BioRender.

FIG 2
FIG 2 Applications of CRISPRi.Pathogenic microbes have many methods of drug resistance and pathogenic strategies (A).CRISPRi has been used across diverse species to investigate these mechanisms.Drug resistance and biofilm formation are particularly represented, given their importance to pathogenesis.CRISPRi/a has also been used successfully to examine other features of fungal pathogenesis (B). Figure was created using BioRender.

TABLE 1
CRISPRi/a applications by species (Continued)